Piggy Back Delivery of Nucleic Acids Into Organisms

ABSTRACT

The present invention includes nucleic acid hybrid molecules capable of entering cells comprising at least one vivo-morpholino oligonucleotide (vivo-MO) comprising a guanidine-rich head conjugated to the 5′ end, and at least one standard oligonucleotide comprising a gene-specific sequence and a standard oligonucleotide pairing sequence, wherein the standard oligonucleotide is bound to the vivo-morpholino oligonucleotide through base pairing to form a hybrid and wherein the vivo-morpholino oligonucleotide pairing sequence is complementary to the standard oligonucleotide pairing sequence.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional application of U.S. patentapplication Ser. No. 13/782,645 filed on Mar. 1, 2013 and entitled“Piggy Back Delivery of Nucleic Acids Into Organisms,” which claimspriority to U.S. Provisional Patent Application Ser. No. 61/605,851,filed Mar. 2, 2012.

TECHNICAL FIELD OF THE INVENTION

Provided are, in general, nucleic acid hybrid molecules capable ofentering cells comprising at least one vivo-morpholino oligonucleotide(vivo-MO) and a standard oligonucleotide comprising a gene-specificsequence; methods of introducing standard oligonucleotides into cells;nucleic acid hybrid molecules for treating patients; and methods ofperforming a clinical trials.

STATEMENT OF FEDERALLY FUNDED RESEARCH

None.

INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC

The present application includes a Sequence Listing filed separately asrequired by 37 CFR 1.821-1.825.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with piggy-back delivery of nucleic acids into organisms.

U.S. Pat. No. 7,935,816 issued to Yong-Fu Li (2011) describespreparations of molecular transporter compositions and their use fortransporting bioactive substances into cells in living animals. For invivo delivery, the composition is covalently linked to the bioactivesubstance and the resultant composite structure is introduced into thesubject. The transporter composition includes multiple guanidinemoieties on a dendrimeric scaffold having a triazine core.

U.S. Patent Application Publication No. 20110190287 to PudurJagadeeswaran (2011) describes compounds comprising a guanidine-richhead covalently coupled to one or more oligonucleotide antisensesequences which are useful to modulate blood coagulation by affectingthe expression of integrin α.IIb or β.3. Included are alsopharmaceutical compositions containing these compounds, with or withoutother therapeutic agents, and well as methods of using these compoundsas inhibitor of platelet aggregation, as thrombolytics, and/or for thetreatment of other thromboembolic disorders. Vivo-MOs, which includeeight guanidine groups dendrimerically arranged in the guanidine-richhead and two synthetic antisense morpholino oligonucleotides, arerepresentative compounds.

U.S. Patent Application Publication No. 20100292306 to George Carlson etal (2010) describes compositions and methods for treatment ofindividuals diagnosed with a dystrophin deficiency are disclosed. Inparticular, inhibitors of NF.K.B transactivation and/or inhibitors thatsuppress p65 expression are used to prevent and/or reverse muscle damagein animals or humans lacking dystrophin. Such compositions and methodsare useful in the treatment of individuals with muscular dystrophy. Inan embodiment of the present disclosure a subject diagnosed withDuchenne muscular dystrophy may be treated with an agent that is aspecific translation blocking vivo-morpholino to decrease the level orthe activity of the p65 subunit of NF.K.B in the muscular tissues of thesubject.

Wu et al., Targeted skipping of human dystrophin exons in transgenicmouse model systemically for antisense drug development, PLoS One. 2011;6(5):e19906. Epub 2011 May 17, describes application of vivo-morpholinoto an hDMD mouse, a transgenic model carrying the full-length humandystrophin gene, and more than 70% efficiency of targeted humandystrophin exon skipping in vivo systemically. We also established aGFP-reporter myoblast culture to screen AOs targeting human dystrophinexon 50. Antisense efficiency for most AOs was consistent between thereporter cells, human myoblasts and in the hDMD mice in vivo. Butvariation in efficiency was also clearly observed.

Nazmi et al, Antiviral and neuroprotective role of octaguanidiniumdendrimer-conjugated morpholino oligomers in Japanese encephalitis, PLoSNegl Trop Dis. 2010 Nov. 23; 4(11):e892.

SUMMARY OF THE INVENTION

The present invention includes nucleic acid hybrid molecule capable ofentering cells comprising at least one vivo-morpholino oligonucleotide(vivo-MO) comprising a guanidine-rich head conjugated to the 5′ end, andat least one standard oligonucleotide comprising a gene-specificsequence and a standard oligonucleotide pairing sequence, wherein thestandard oligonucleotide is bound to the vivo-morpholino oligonucleotidethrough base pairing to form a hybrid, and wherein the vivo-morpholinooligonucleotide pairing sequence is complementary to the standardoligonucleotide pairing sequence. In certain aspects of the invention,the gene-specific sequence comprises a sequence that is antisense to anmRNA or a pre-mRNA and/or is complementary to at least one coding DNA,noncoding DNA, or a splice site; the standard oligonucleotide maycomprise a DNA oligonucleotide, a RNA, a siRNA or a RNAi, and/or a gene.In certain aspects of the invention, the standard oligonucleotide may beselected from the group consisting of phosphorodithio oligonucleotide,phosphorothio oligonucleotide, locked oligonucleotide, and peptidenucleic acid. The standard oligonucleotide pairing sequence may belocated 3′ of the gene-specific sequence, whereby the standardoligonucleotide has a 5′ overhanging end of 20-30 nucleotides. Incertain aspects, the morpholino oligonucleotide pairing sequence and thegene-specific oligonucleotide pairing sequence may be 12-20 long; theguanidine-rich head may comprise a dendrimeric octaguinidine.Embodiments of the invention includes methods of introducing a standardoligonucleotide into a cell comprising obtaining a standardoligonucleotide comprising a gene-specific sequence and a standardoligonucleotide pairing sequence, obtaining a vivo-morpholinooligonucleotide (vivo-MO) comprising a guanidine-rich head conjugated tothe 5′ end and a vivo-morpholino oligonucleotide pairing sequence thatis complementary to the standard oligonucleotide pairing sequence,binding the standard oligonucleotide to the vivo-morpholinooligonucleotide, wherein a nucleic acid hybrid molecule is formed, andcontacting a cell with the nucleic acid hybrid molecule. The cell mayhave been previously isolated and contacting the cell with the nucleicacid hybrid molecule may be selected from the group consisting ofadministering the nucleic acid hybrid molecule to a vertebrate orally,intravenously, intramuscularly, intraperitoneally, subcutaneously, byintranasal instillation, by application to mucous membranes, and byinstillation into hollow organ walls or newly vascularized bloodvessels. Contacting the cell with the nucleic acid hybrid molecule mayalso comprise treating a patient afflicted with a disease selected fromthe group consisting of diabetes, cancer, thalassemia, sickle celldisease, hemophilia, viral hepatitis, AIDS, genetic disease, andinfectious disease. In certain aspects, the method may further comprisedetermining a gene expression of a gene complementary to thegene-specific sequence, and the standard oligonucleotide may be selectedfrom the group consisting of a DNA oligonucleotide, a RNA, a RNAi, asiRNA, phosphorodithio oligonucleotide, a phosphorothio oligonucleotide,a locked oligonucleotide, and a peptide nucleic acid. The standardoligonucleotide may comprise a gene, and the guanidine-rich head maycomprise a dendrimeric octaguinidine. The present invention alsoincludes methods to treat a patient suspected of having a diseasecomprising obtaining a nucleic acid hybrid molecule capable of enteringcells comprising at least one standard oligonucleotide comprising agene-specific sequence and a standard oligonucleotide pairing sequence,at least one vivo-morpholino oligonucleotide (vivo-MO) comprising aguanidine-rich head conjugated to the 5′ end, wherein thevivo-morpholino oligonucleotide pairing sequence is complementary to thestandard oligonucleotide pairing sequence, and wherein the standardoligonucleotide is bound to the vivo-morpholino oligonucleotide throughbase pairing, forming a hybrid; and contacting the patient with thenucleic acid hybrid molecule. The methods may also comprise obtaining asample from the patient and determining gene expression of a gene thatis homologous, antisense, or complementary to the gene-specificsequence. The disease may be selected from the group consisting ofdiseases treatable by reducing a translation of a gene, wherein the geneis a c-myc gene and the disease is Burkitt's lymphoma or the gene is aras oncogene and the disease is adenocarcinomas, a thyroid tumor, or apancreatic tumor, and contacting the patient with the nucleic acidhybrid molecule may be selected from the group consisting ofadministering the nucleic acid hybrid molecule orally, intravenously,intramuscularly, intraperitoneally, subcutaneously, by intranasalinstillation, by application to mucous membranes, and by instillationinto hollow organ walls or newly vascularized blood vessels. In certainaspects, the standard oligonucleotide is selected from the groupconsisting of a DNA oligonucleotide, a RNA, a RNAi, a siRNA, aphosphorodithio oligonucleotide, a phosphorothio oligonucleotide, alocked oligonucleotide, and a peptide nucleic acid, and the nucleic acidhybrid molecule may comprise a standard oligonucleotide comprising agene-specific sequence and a standard oligonucleotide pairing sequence,a vivo-morpholino oligonucleotide (vivo-MO) comprising a guanidine-richhead conjugated to the 5′ end and a vivo-morpholino oligonucleotidepairing sequence that is complementary to the standard oligonucleotidepairing sequence, the standard oligonucleotide bound to thevivo-morpholino oligonucleotide through base pairing, forming a hybridnucleic acid. In certain aspects, the disease may be selected from thegroup consisting of diabetes, cancer, genetic disorder, diabetes,infectious disease, hemophilia, thalassemia, sickle cell disease, andDuchene Muscular dystrophy etc. In certain embodiments, the inventionincludes methods of performing a clinical trial to evaluate a candidatedrug believed to be useful in treating a disease by affecting theexpression of a gene complementary, antisense, or homologous to agene-specific sequence, the method comprising: (a) obtaining thecandidate drug comprising a nucleic acid hybrid molecule capable ofentering cells comprising at least one standard oligonucleotidecomprising the gene-specific sequence and a standard oligonucleotidepairing sequence, at least one vivo-morpholino oligonucleotide (vivo-MO)comprising a guanidine-rich head conjugated to the 5′ end; wherein thevivo-morpholino oligonucleotide pairing sequence is complementary to thestandard oligonucleotide pairing sequence; and wherein the standardoligonucleotide is bound to the vivo-morpholino oligonucleotide throughbase pairing, forming a hybrid (b) administering the candidate drug to afirst subset of patients, and a placebo to a second subset of patients;a comparable drug to a second subset of patients; or a drug combinationof the candidate drug and another active agent to a second subset ofpatients; (c) monitoring the progression of the disease in the firstsubset of patients as compared to the second subset of patients, whereina relative and statistically significant improvement of the disease inthe first subset of patients indicates that the candidate drug is usefulin treating the disease. In certain embodiments, the invention includesmethods to identify a nucleic acid hybrid molecule effective in reducingan expression of a gene comprising: obtaining a first and at least asecond nucleic acid hybrid molecule, each nucleic acid hybrid moleculecomprising at least one standard oligonucleotide comprising a sequencespecific for the gene and a standard oligonucleotide pairing sequence;each nucleic acid hybrid molecule further comprising at least onevivo-morpholino oligonucleotide (vivo-MO) comprising a guanidine-richhead conjugated to the 5′ end, wherein the vivo-morpholinooligonucleotide pairing sequence is complementary to the standardoligonucleotide pairing sequence, and wherein the standardoligonucleotide is bound to the vivo-morpholino oligonucleotide throughbase pairing, forming a hybrid, contacting the first and at least thesecond nucleic acid hybrid molecules with at least one cell each; anddetermining the expression of the gene in the cells contacted with thefirst and at least the second nucleic acid hybrid molecules; identifyingthe nucleic acid hybrid molecules effective in reducing the expressionof the gene. In certain aspects, the methods further comprisedetermining the expression of a control gene and identifying the nucleicacid hybrid molecule that does not affect the expression of the controlgene. In certain embodiments, the invention includes methods to reducean expression of a gene comprising obtaining a standard oligonucleotidecomprising a gene-specific sequence and a standard oligonucleotidepairing sequence, obtaining a vivo-morpholino oligonucleotide (vivo-MO)comprising a guanidine-rich head conjugated to the 5′ end and avivo-morpholino oligonucleotide pairing sequence that is complementaryto the standard oligonucleotide pairing sequence, binding the standardoligonucleotide to the vivo-morpholino oligonucleotide, wherein anucleic acid hybrid molecule is formed, and contacting at least one cellwith the nucleic acid hybrid molecule, whereby the hybrid moleculeenters the cell and whereby the nucleic acid hybrid molecule reduces theexpression of the gene. Contacting the cell with the nucleic acid hybridmolecule may be selected from the group consisting of administering thenucleic acid hybrid molecule orally, intravenously, intramuscularly,intraperitoneally, subcutaneously, by intranasal instillation, byapplication to mucous membranes, and by instillation into hollow organwalls or newly vascularized blood vessels. In addition, contacting thecell with the nucleic acid hybrid molecule may comprise treating apatient afflicted with a disease selected from the group consisting ofdiabetes, cancer, genetic disease, and infectious disease. In certainaspects, the standard oligonucleotide may be selected from the groupconsisting of a DNA oligonucleotide, a RNA, a RNAi a siRNA, aphosphorodithio oligonucleotide, a phosphorothio oligonucleotide, alocked oligonucleotide, and a peptide nucleic acid, and/or theguanidine-rich head may comprise a dendrimeric octaguinidine.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures.

FIG. 1 is a schematic diagram of the hybrid formation. Note the possiblehybrid formation (base pairing indicated by small vertical bars) betweennon-gene Vivo-Morpholino, ngVMO (shown in green) and standardoligonucleotide, SO (shown in blue) as well as hybrid formation betweenSO and either mRNA or pre-mRNA (shown in red). Arrows show RNaseHcleavage of the mRNA/pre-mRNA portion of the RNA-DNA hybrid. Closedcircle represents dendrimeric octaguinidine conjugated at the 5′ end ofngVMO.

FIG. 2 shows αIIb-SO/ngVMO hybrid induced alternative splicing resultsin deletion of exon 20. Agarose gel showing the RT-PCR products fromwhite cells including thrombocytes prepared from adult zebrafishinjected with control SO hybrid (Control) and αIIb-SO/ngVMO hybrid(αIIb). Arrows show bands corresponding to the normal splice product(396 bp) and alternatively spliced product (149 bp). 2-log DNA Ladder(New England Biolabs, Ipswich, Mass.) used as DNA size markers (Marker)are in left lane.

FIG. 3 shows gill-bleeding assay: Fish were photographed and the numberof red pixels representing the bleeding were measured in control SOhybrid treated (Control) and αIIb-SO/ngVMO hybrid (αIIb) treatedzebrafish (N=6). p value is <0.001 between the Control and αIIb.

FIGS. 4 a-4 b shows blood smear from TG(fli1:EGFP)y1 transgenic fishtreated with EGFP-SO/ngVMO hybrid. Figures a and b are the brightfieldand fluorescence images respectively. Note that there are severalthrombocytes lacking GFP fluorescence shown by black arrows in 4 a. Onlya few thrombocytes are GFP positive shown by red arrows in 4 a. Whitearrows in 4 b show the GFP thrombocytes.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

Knockdown of genes is used to establish the function of genes in animalmodels as well as in cells. However, application of these methods on agenome wide basis for all the genes (approximately 30,000 genes) has notbeen accomplished because it is prohibitively expensive; for exampleperforming one knockdown costs approximately $1000. Furthermore,delivering the reagents to cells is also complex and sometimes resultsin poor efficiencies and toxicities. Knockdowns have been achieved bythe use of antisense oligonucleotides and siRNA, e.g., standardoligonucleotides and modified oligonucleotides (MOs and Vivo-MOs as wellas phosphorothionates). The inventors recognize that MOs and Vivo-MOscan be used to inhibit gene functions because these MOs are more stableas they are not degraded by nucleases, unlike the standardoligonucleotides. The advantage of Vivo-MOs is that they penetrate thecells and therefore one can deliver them to cells without using specialtechniques such as microinjection or chemical delivery methods.

The inventors recognized that hybrid molecule can be formed betweenVivo-MO and a nucleic acid (standard oligonucleotide) by base pairing,and the hybrid molecule is capable of entering a cell along with thecell penetrating Vivo-MO.

In one example and non-limiting embodiment, the nucleic acid hybridmolecule has the ability to block a gene involved in platelet function,e.g., as a non-limiting model system, zebrafish. In another workingmodel, example, and non-limiting embodiment, the present inventorsemployed a reagent to target fish whose thrombocytes are labeled withGFP and affirmed loss of GFP signal in thrombocytes. Interestingly toconduct one knockdown by this method is currently only $10. Thus, 20,000gene knockdowns can be performed easily and in a cost-effective manner.Such principle will lead to the establishment of functions of novelgenes in any desired pathway for example in thrombosis, diabetes, heartdisease and cancer.

A schematic diagram of an embodiment of a nucleic acid hybrid molecule:

The top strand is the Vivo Morpholino (e.g., 25 nt). * denotes themodification which facilitates the entry of the Vivo-MO. Dots representbase pairing (e.g., 14 bp). The bottom strand standard oligonucleotide.The unpaired region (e.g., 25 nt) of the standard oligonucleotidecomprises a gene-specific sequence, e.g., a region that is complimentaryto the mRNA of interest, e.g., the target for knockdown.

The knockdown of protein functions by antisense oligonucleotides hasbeen used to understand protein function. The inventors recognize thatVivo-Morpholinos (Vivo-MOs), chemically modified morpholinos that canpenetrate cells, can be used in adult experimental animal models toalter pre-mRNA splicing or to block mRNA translation and thereby changethe protein expression, e.g., the present inventors have injectedthrombocyte-specific αIIb Vivo-MOs intravenously and inhibitedthrombocyte function in adult zebrafish. In addition to Vivo-MOs, otherantisense oligonucleotides can be used to knockdown protein expressionby taking advantage of the endogenous RNaseH mechanism which cleavestarget mRNA.

Here, the present inventors introduce nucleic acid hybrid molecules madeof a non-gene specific Vivo-MO (ngVMO) and a gene specific standardoligonucleotide (SO). In one embodiment, the hybrid is designed to basepair leaving 5′ overhanging ends. The unbasepaired gene specific SO is,at least partially, antisense to the target mRNA/premRNA. The hybridmolecule has the ability to enter cells because the SO is piggy-backedonto the ngVMO. The present inventors have validated this concept bytargeting two proteins, αIIb and EGFP, by using the above piggy-backingstrategy and found that expression of both proteins was effectivelyreduced, resulting in both increased bleeding and loss of EGFP. Thisapproach is less expensive and more efficient compared to a Vivo-MOknockdown. Therefore, these nucleic acid hybrid molecules makelarge-scale functional genomics a realistic goal rather than amultimillion-dollar undertaking. This approach can be used to inhibitundesired protein expression in a variety of human disorders includingAIDS. Furthermore, since RNAs and genes can be piggy-backed usingngVMOs, in one embodiment, the present nucleic acid hybrid molecules canbe used as gene therapy in cases when protein expression is desirable.

The inventors recognize that antisense oligonucleotides can be used toknockdown protein levels by either translational blocking or spliceblocking to control cancer and viral infections with the goal oftreating human diseases [1; 2]. This knockdown inhibition can be used inmodel organisms, such as zebrafish, predominantly through the use ofmorpholino oligonucleotides (MOs), to study functions of proteins inboth development and disease, particularly as a gene discovery tool [3;4]. These MOs are introduced into the yolks of 1-8-cell-stage zebrafishembryos. Due to the cytoplasmic bridges, MOs rapidly diffuse into thesecells allowing ubiquitous cytosolic delivery. However, direct cytosolicdelivery of MOs into cells has been difficult to achieve with theexception of microinjections. Photoactivatable MOs can be introduced toachieve tissue-specific knockdowns in embryos [5]. And conjugation ofdendrimeric octaguinidine to MOs (Vivo-MOs) results in permeability ofMOs into cells [6]. Because of this membrane diffusible nature and lackof toxicity, use in human therapy is possible [7]. And Vivo-MOs can beemployed to evaluate their use in treatment of Duchenne musculardystrophy [8].

The present inventors utilized Vivo-MO technology to inhibit thrombocytefunction in adult zebrafish for the first time by injectingthrombocyte-specific αIIb Vivo-MOs intravenously into adult zebrafish;thereby, established proof of principle and provided the basis to targettwo other novel candidate thrombocyte receptors, to knockdownthrombocyte function, and to evaluate the function of novel genesinvolved in, e.g., hemostatic pathways. The present inventors have shownthat these novel receptors are present in human platelets and thereforethis finding is applicable to human platelet function.

The present inventors recognize that currently available antisenseoligonucleotide technologies include inhibition of protein synthesis bythe siRNA, standard oligonucleotides (SOs), and modifiedoligonucleotides such as, phosphorothio oligonucleotide, lockedoligonucleotides, and peptide nucleic acids. In all these approaches,delivery of the oligonucleotides into cells requires complexing theseoligonucleotides with PEI or calcium phosphate precipitations. Thismethodology, however, cannot be used on whole organisms due to toxicityissues. The present inventors acknowledge that SOs are considered to bemore toxic than phosphorothio nucleotides. In addition, when theseantisense oligonucleotides are base-paired with the target mRNAs, themRNAs are cleaved by RNaseH, and, therefore, appear to be more efficientthan translational blocking or splice blocking induced by the morpholinooligonucleotides.

The present inventors recognize that if an SO is synthesized such thatit is complementary to the target mRNA/pre-mRNA on the 5′ side andcomplementary to a non-gene specific Vivo-MO (ngVMO) on the 3′ side(FIG. 1), then this SO/ngVMO hybrid will enter the cell since the SO is‘piggy-backed’ onto the ngVMO. Once inside the cell, in one embodiment,the SO portion binds to the target mRNA/pre-mRNA and leads to thecleavage of the target mRNA by the endogenous RNaseH mechanism. In otherembodiments, the SO participates in splice and/or translation blockingusing a mechanism similar to that of Vivo-MO targeting.

The present inventors demonstrate that in one non-limiting example orembodiment, a splice blocking SO specific for αIIb piggy-backed withngVMO (αIIb-SO/ngVMO) can be used to inhibit thrombocyte function inadult zebrafish by intravenous injection. Furthermore, an SO specificfor EGFP piggy-backed with ngVMO (EGFP-SO/ngVMO) can be used to inhibitEGFP in zebrafish thrombocytes. This targeting is not only efficient butalso cost effective when compared to Vivo-MO targeting [9].

Zebrafish SO/ngVMO injections to generate knockdowns: A ngVMO5′-CCTCTTACCTCAGTTACAATTTATA-3′ (SEQ ID NO: 1) was purchased fromGene-Tools LLC, Philomath Oreg. A SO was designed so that it canhybridize both to ngVMO (14 bp) and to αIIb pre-mRNA at the donor splicesite of exon 20 (25 bp), 5′-GGAAGTGACTAAACCCTCACCTCATTATAAATTGTAACTG-3′(SEQ ID NO: 2). A control SO that can hybridize to ngVMO and has acomplementary sequence corresponding to the antisense sequence portionof the above SO, 5′-ATGAGGTGAGGGTTTAGTCACTTCCTATAAATTGTAACTG-3′ (SEQ IDNO: 3) was designed. Two other SOs were designed: one that targets EGFPmRNA and the other its control,5′-TGTACATAACCTTCGGGCATGGCACTATAAATTGTAACTG-3′ (SEQ ID NO: 4) and5′-GTGCCATGCCCGAAGGTTATGTACATATAAATTGTAACTG-3′ (SEQ ID NO: 5),respectively. All SOs and their controls were purchased from Invitrogen,Carlsbad, Calif. 4.5 μl of 0.5 mM ngVMO was mixed with 4.5 μl of 0.5 mMSO and 1 μl 10× phosphate buffered saline, pH 7.4 (PBS). The mixture washeated at 90 degree C. and slowly cooled to room temperature so that theSO and ngVMO could hybridize. 5 μl of this hybridized SO/ngVMO was usedto inject an adult zebrafish intravenously. αIIb-SO/ngVMO hybrid wasinjected into wild type zebrafish whereas EGFP-SO/ngVMO hybrid wasinjected into TG(fli1:EGFP)y1 zebrafish, which carries the transgene ofthe FLI1 gene promoter driving GFP and in which all thrombocytes are GFPpositive.

Thrombocyte functional gill bleeding assay: Gill bleeding was induced byplacing the fish in a petridish containing 50 ml of 50 μM NaOH. The fishwere anesthetized in 50 ml of 2 mM tricaine (Sigma-Aldrich, St. Louis,Mo.) for 3 minutes prior to placing them in NaOH. The fish werephotographed with a Nikon E995 Coolpix camera and the red pixels werecounted by Adobe Photoshop software 7.0 and used to quantify bleeding.

RT-PCR: Zebrafish blood was centrifuged at 500 g and the white celllayer was used in the cell to cDNA kit (Agilent Technologies, LaJolla,Calif.) to amplify the αIIb mRNA. The present inventors designed forward5′-AGTGCTGCATGGACAAAGTG-3′ (SEQ ID NO: 6) and reverse5′-GGTTCTCCACCTGTTCCAGA-3′ (SEQ ID NO: 7) primers for exons 18 and 22,respectively; these were synthesized by Biosynthesis, Lewisville, Tex.These primers were used to amplify the 396 bp product. In the case ofexon skipping, the predicted product is 149 base pair. These RT-PCRproducts were resolved on 1.5% agarose gels.

GFP fluorescence of thrombocytes: GFP fluorescence was detected onfreshly prepared blood smears from EGFP-SO/ngVMO hybrid injectedTG(fli1:EGFP)y1 transgenic zebrafish and was photographed forimmunofluorescence using a Nikon Eclipse 80i microscope.

Statistical analysis: Statistical analysis was performed using SigmaPlot 10 with Sigma Stat integration software. Statistical significancewas assessed by ANOVA and a p value <0.05 was considered significant.

To inhibit the synthesis of αIIb by knockdown method, the presentinventors chose to splice out exon 20 of αIIb pre-mRNA; the presentinventors have also targeted this exon in work using Vivo-MO specificfor αIIb [9]. 5 μl αIIb-SO/ngVMO hybrid were injected intravenously intoadult zebrafish and, after 24 hrs, RNA was isolated from white celllayer which contains thrombocytes. This RNA was analyzed for alternativesplicing by using primers designed from exon 18 and 22 on RNA preparedfrom these thrombocytes. If normal splicing occurs, a 396 bp product isgenerated. If exon skipping occurs, a 149 bp of DNA is generated. A 149bp band was obtained in the thrombocytes of zebrafish whereαIIb-SO/ngVMO hybrid was injected compared to control SO hybrid (FIG.2). Furthermore, the overall intensity of the 396 bp band was reducedand, in fact, in some experiments the band in this region was completelynonexistent.

Gill-bleeding assay was performed to show that the reduction in mRNAproducing αIIb also result in greater bleeding. The fish treated withαIIb-SO/ngVMO hybrid exhibited more bleeding compared to the fishtreated with control hybrid (FIG. 3).

EGFP synthesis was inhibited in thrombocytes that are GFP+ by injectingEGFP-SO/ngVMO hybrid intravenously into TG(fli1:EGFP)y1 transgeniczebrafish. Twenty-four hours after injection, a blood smear was preparedfrom these transgenic fish and GFP fluorescence in thrombocytes wasobserved: Almost 70-80% of thrombocytes lost GFP fluorescence (FIGS. 4 aand 4 b).

The present inventors demonstrate that, as a non-limiting example,direct injection of αIIb-SO/ngVMO hybrid intravenously into zebrafishinhibits αIIb and thus reduces thrombocyte aggregation. The RT-PCRresults show that alternatively spliced αIIb mRNA is generated resultingin a 149 bp product (confirmed by sequence analysis) which providesevidence that the αIIb-SO/ngVMO hybrid is effectively penetratingthrombocytes. Interestingly, the effect of αIIb-SO/ngVMO hybrid onthrombocyte aggregation was also observed in 24 hrs. This result issimilar to what the inventors observed with αIIb Vivo-MO [9]. However,the overall cumulative intensities of the 396 bp and 149 bp bands werereduced when compared to the 396 bp band in controls. This documentsthat, in addition to the alternative splicing due to splice blocking,the pre-mRNA is cleaved by RNaseH. Therefore, the EGFP study wasconducted: SO were used that targets the EGFP mRNA sequence locatedapproximately in the middle of translational initiation and terminatorcodons. Thus, the target is neither a translational blocker nor a spliceblocker. Thus, reduction in EGFP in thrombocyte is due to thedegradation of EGFP RNA by RNaseH mechanism. The results demonstrated70-80% GFP− thrombocytes showing that the EGFP-SO/ngVMO hybrid method isvery efficient and degrades EGFP RNA by RNaseH mechanism.

Because thrombocytes are readily accessible, the inhibition of αIIb inadult zebrafish provides therapeutic possibilities, provides the abilityto inhibit the function of any thrombocyte specific gene and performbiochemical studies, and serves as a example of the predictability andfeasibility to generally employ this methodology to other genes andother sequences. The present inventors recognize that, for example,thrombocyte surface receptors such as ADP receptors, thromboxanereceptors, other GPCRs, and several signaling molecules, such askinases, can be inhibited. Thus, this method has the advantage ofinhibiting proteins for which small molecule inhibitors are notcurrently available. Furthermore, because of the ease of injections andthrombocyte assays, large-scale, high-throughput, genome-wide knockdownscan be designed to identify novel genes participating in thrombocytedevelopment and function. In addition, the same principles can be usedto understand other hematological disorders as well as disorders thatare amenable for studies by simple injections of these hybrid SOs. Suchgenome-wide knockdowns are possible because SOs are significantly lessexpensive than MOs or Vivo-MOs. The present inventors recognize thatsome off-target knockdowns and toxicity are both inherent problemscommon to all antisense technologies. Nevertheless, after initial,large-scale screening, the toxic effects can be resolved by using lesstoxic phosphorothio oligonucleotides, which can also form hybridssimilar to SOs, and off-target knockdown issues have to be confirmed byeither genome TILLING or mutagenesis methods.

The present inventors have demonstrated that it is possible to inhibitthrombocyte specific function by exon skipping using αIIb-SO/ngVMOhybrids, and GFP expression can be eliminated from GFP+ thrombocytes inadult zebrafish by using EGFP-SO/ngVMO hybrids. This proof of principleof inhibition of protein expression in adult zebrafish by SO hybrids hastremendous applicability, not only to identify functions of novel genesin thrombocytes but also in other accessible blood cells as well as inhighly vascular organs. Since it is possible to deliver SO hybrids toany hematopoietic cells, it is possible to use these reagents not onlyas an antithrombotic agent but also as an agent to correct otherhematological disorders including AIDS by degrading HIV RNA. Theefficiency of knockdowns can be increased by designing multiple SOs forthe same target mRNA. Furthermore, by conducting random knockdowns ofmultiple protein functions, complex genetic disorders, such as diabetes,can be created in model organisms. In this way, the mechanisms of theseand many other disorders come to light. In addition, RNAs and genes canbe delivered easily into cells by complexing with the ngVMO, thus, thistechnology is applicable to gene therapy as well as RNA/proteintherapies. Furthermore, efficient delivery of siRNA piggy-backed onngVMO into the cells in whole organism should also be feasible. Thisallows for an additional, complementary approach to inhibit proteinexpression. Thus, the present technology has numerous applications.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

-   [1] R. M. Hudziak, J. Summerton, D. D. Weller, and P. L. Iversen,    Antiproliferative effects of steric blocking phosphorodiamidate    morpholino antisense agents directed against c-myc. Antisense    Nucleic Acid Drug Dev 10 (2000) 163-76.-   [2] C. S. Cobbs, T. R. Whisenhunt, D. R. Wesemann, L. E.    Harkins, E. G. Van Meir, and M. Samanta, Inactivation of wild-type    p53 protein function by reactive oxygen and nitrogen species in    malignant glioma cells. Cancer Res 63 (2003) 8670-3.-   [3] A. Nasevicius, and S. C. Ekker, Effective targeted gene    ‘knockdown’ in zebrafish. Nat Genet 26 (2000) 216-20.-   [4] M. Gregory, R. Hanumanthaiah, and P. Jagadeeswaran, Genetic    analysis of hemostasis and thrombosis using vascular occlusion.    Blood Cells Mol Dis 29 (2002) 286-95.-   [5] I. A. Shestopalov, S. Sinha, and J. K. Chen, Light-controlled    gene silencing in zebrafish embryos. Nat Chem Biol 3 (2007) 650-1.-   [6] P. A. Morcos, Y. Li, and S. Jiang, Vivo-Morpholinos: a    non-peptide transporter delivers Morpholinos into a wide array of    mouse tissues. Biotechniques 45 (2008) 613-4, 616, 618 passim.-   [7] J. D. Moulton, and S. Jiang, Gene knockdowns in adult animals:    PPMOs and vivo-morpholinos. Molecules 14 (2009) 1304-23.-   [8] B. Wu, Y. Li, P. A. Morcos, T. J. Doran, P. Lu, and Q. L. Lu,    Octa-guanidine morpholino restores dystrophin expression in cardiac    and skeletal muscles and ameliorates pathology in dystrophic mdx    mice. Mol Ther 17 (2009) 864-71.-   [9] S. Kim, U. P. Radhakrishnan, S. K. Rajpurohit, V. Kulkarni,    and P. Jagadeeswaran, Vivo-Morpholino knockdown of alphaIIb: A novel    approach to inhibit thrombocyte function in adult zebrafish. Blood    Cells Mol Dis 44 169-74.

SEQUENCE LISTING SEQ ID NO: 1 CCTCTTACCTCAGTTACAATTTATA SEQ ID NO: 2GGAAGTGACTAAACCCTCACCTCATTATAAATTGTAACTG SEQ ID NO: 3ATGAGGTGAGGGTTTAGTCACTTCCTATAAATTGTAACTG SEQ ID NO: 4TGTACATAACCTTCGGGCATGGCACTATAAATTGTAACTG SEQ ID NO: 5GTGCCATGCCCGAAGGTTATGTACATATAAATTGTAACTG SEQ ID NO: 6AGTGCTGCATGGACAAAGTG SEQ ID NO: 7 GGTTCTCCACCTGTTCCAGA

What is claimed is:
 1. A nucleic acid hybrid molecule capable ofentering cells comprising: at least one vivo-morpholino oligonucleotide(vivo-MO) comprising a guanidine-rich head at the 5′ end; and at leastone oligonucleotide comprising a gene-specific sequence and aoligonucleotide pairing sequence, wherein the oligonucleotide is boundto the vivo-morpholino oligonucleotide through base pairing to form ahybrid; and wherein the vivo-morpholino oligonucleotide pairing sequenceis complementary to the oligonucleotide pairing sequence. 2.-4.(canceled)
 5. The nucleic acid hybrid molecule of claim 1, wherein theoligonucleotide comprises a RNA.
 6. The nucleic acid hybrid molecule ofclaim 1, wherein the oligonucleotide comprises a siRNA or a RNAi. 7.-11.(canceled)
 12. A method of introducing a oligonucleotide into a cellcomprising: obtaining a oligonucleotide comprising a gene-specificsequence and a oligonucleotide pairing sequence; obtaining avivo-morpholino oligonucleotide (vivo-MO) comprising a guanidine-richhead conjugated to the 5′ end and a vivo-morpholino oligonucleotidepairing sequence that is complementary to the oligonucleotide pairingsequence; binding the oligonucleotide to the vivo-morpholinooligonucleotide, wherein a nucleic acid hybrid molecule is formed; andcontacting a cell with the nucleic acid hybrid molecule.
 13. The methodof claim 12, wherein the cell has been previously isolated.
 14. Themethod of claim 12, wherein contacting the cell with the nucleic acidhybrid molecule is selected from the group consisting of administeringthe nucleic acid hybrid molecule to a vertebrate orally, intravenously,intramuscularly, intraperitoneally, subcutaneously, by intranasalinstillation, by application to mucous membranes, and by instillationinto hollow organ walls or newly vascularized blood vessels.
 15. Themethod of claim 12, wherein contacting the cell with the nucleic acidhybrid molecule comprises treating a patient afflicted with a diseaseselected from the group consisting of diabetes, cancer, thalassemia,sickle cell disease, hemophilia, viral hepatitis, AIDS, genetic disease,and infectious disease.
 16. The method of claim 12, further comprisingdetermining a gene expression of a gene complementary to thegene-specific sequence.
 17. The method of claim 12, wherein theoligonucleotide is selected from the group consisting of a DNAoligonucleotide, a RNA, a RNAi, a siRNA, phosphorodithiooligonucleotide, a phosphorothio oligonucleotide, a lockedoligonucleotide, and a peptide nucleic acid.
 18. The method of claim 12,wherein the oligonucleotide comprises a gene.
 19. The method of claim12, wherein the guanidine-rich head comprises a dendrimericoctaguinidine.
 20. A method to treat a patient suspected of having adisease comprising: obtaining a nucleic acid hybrid molecule capable ofentering cells comprising at least one oligonucleotide comprising agene-specific sequence and a oligonucleotide pairing sequence, at leastone vivo-morpholino oligonucleotide (vivo-MO) comprising aguanidine-rich head conjugated to the 5′ end, wherein thevivo-morpholino oligonucleotide pairing sequence is complementary to theoligonucleotide pairing sequence, and wherein the oligonucleotide isbound to the vivo-morpholino oligonucleotide through base pairing,forming a hybrid; and contacting the patient with the nucleic acidhybrid molecule.
 21. The method of claim 20, further comprising:obtaining a sample from the patient; and determining gene expression ofa gene that is homologous, antisense, or complementary to thegene-specific sequence.
 22. The method of claim 20, wherein the diseaseis selected from the group consisting of diseases treatable by reducinga translation of a gene, wherein the gene is a c-myc gene and thedisease is Burkitt's lymphoma or the gene is a ras oncogene and thedisease is adenocarcinomas, a thyroid tumor, or a pancreatic tumor. 23.The method of claim 20, wherein contacting the patient with the nucleicacid hybrid molecule is selected from the group consisting ofadministering the nucleic acid hybrid molecule orally, intravenously,intramuscularly, intraperitoneally, subcutaneously, by intranasalinstillation, by application to mucous membranes, and by instillationinto hollow organ walls or newly vascularized blood vessels.
 24. Themethod of claim 20, wherein the oligonucleotide is selected from thegroup consisting of a DNA oligonucleotide, a RNA, a RNAi, a siRNA, aphosphorodithio oligonucleotide, a phosphorothio oligonucleotide, alocked oligonucleotide, and a peptide nucleic acid. 25.-26. (canceled)27. A method of performing a clinical trial to evaluate a candidate drugbelieved to be useful in treating a disease by affecting the expressionof a gene complementary, antisense, or homologous to a gene-specificsequence, the method comprising: (a) obtaining the candidate drugcomprising a nucleic acid hybrid molecule capable of entering cellscomprising at least one oligonucleotide comprising the gene-specificsequence and a oligonucleotide pairing sequence; at least onevivo-morpholino oligonucleotide (vivo-MO) comprising a guanidine-richhead conjugated to the 5′ end; wherein the vivo-morpholinooligonucleotide pairing sequence is complementary to the oligonucleotidepairing sequence; and wherein the oligonucleotide is bound to thevivo-morpholino oligonucleotide through base pairing, forming a hybrid;(b) administering the candidate drug to a first subset of patients, anda placebo to a second subset of patients; a comparable drug to a secondsubset of patients; or a drug combination of the candidate drug andanother active agent to a second subset of patients; and (c) monitoringthe progression of the disease in the first subset of patients ascompared to the second subset of patients, wherein a relative andstatistically significant improvement of the disease in the first subsetof patients indicates that the candidate drug is useful in treating thedisease.
 28. A method to identify a nucleic acid hybrid moleculeeffective in reducing an expression of a gene comprising: obtaining afirst and at least a second nucleic acid hybrid molecule, each nucleicacid hybrid molecule comprising at least one oligonucleotide comprisinga sequence specific for the gene and a oligonucleotide pairing sequence;each nucleic acid hybrid molecule further comprising at least onevivo-morpholino oligonucleotide (vivo-MO) comprising a guanidine-richhead conjugated to the 5′ end, wherein the vivo-morpholinooligonucleotide pairing sequence is complementary to the oligonucleotidepairing sequence, and wherein the oligonucleotide is bound to thevivo-morpholino oligonucleotide through base pairing, forming a hybrid,contacting the first and at least the second nucleic acid hybridmolecules with at least one cell each; determining the expression of thegene in the cells contacted with the first and at least the secondnucleic acid hybrid molecules; and identifying the nucleic acid hybridmolecules effective in reducing the expression of the gene.
 29. Themethod of claim 28 further comprising: determining the expression of acontrol gene; and identifying the nucleic acid hybrid molecule that doesnot affect the expression of the control gene.
 30. A method to reduce anexpression of a gene comprising: obtaining a oligonucleotide comprisinga gene-specific sequence and a oligonucleotide pairing sequence;obtaining a vivo-morpholino oligonucleotide (vivo-MO) comprising aguanidine-rich head conjugated to the 5′ end and a vivo-morpholinooligonucleotide pairing sequence that is complementary to theoligonucleotide pairing sequence; binding the oligonucleotide to thevivo-morpholino oligonucleotide, wherein a nucleic acid hybrid moleculeis formed; and contacting at least one cell with the nucleic acid hybridmolecule, whereby the hybrid molecule enters the cell and whereby thenucleic acid hybrid molecule reduces the expression of the gene.
 31. Themethod of claim 30, wherein contacting the cell with the nucleic acidhybrid molecule is selected from the group consisting of administeringthe nucleic acid hybrid molecule orally, intravenously, intramuscularly,intraperitoneally, subcutaneously, by intranasal instillation, byapplication to mucous membranes, and by instillation into hollow organwalls or newly vascularized blood vessels.
 32. The method of claim 30,wherein contacting the cell with the nucleic acid hybrid moleculecomprises treating a patient afflicted with a disease selected from thegroup consisting of diabetes, cancer, genetic disease, and infectiousdisease.
 33. The method of claim 30, wherein the oligonucleotide isselected from the group consisting of a DNA oligonucleotide, a RNA, aRNAi a siRNA, a phosphorodithio oligonucleotide, a phosphorothiooligonucleotide, a locked oligonucleotide, and a peptide nucleic acid.34. The method of claim 30, wherein the guanidine-rich head comprises adendrimeric octaguinidine.